In Depth Characterization of the Promoter Proximal Proteome of Single Copy Locus FOXP2

Identifying the proteins that interact with sequence-defined chromatin segments is a critical step in understanding gene expression. Most procedures perform bulk analysis of samples to identify the general interactions that occur in a cellular population and thereby do not detect the proteins that operate at a single locus. To circumvent this limitation, we developed a modified method that uses genetically targeted proximity labeling with dCas9-APEX2 to specifically biotinylate the promoter proximal proteome of the single copy locus FOXP2 in live HEK293 cells. After capture of the tagged proteins with streptavidin and isobaric labeling of the peptides produced from on-bead digestion with tandem mass tags, we used quantitative 2D-LC-MS3 on a tribrid mass spectrometer to identify 373 significantly enriched proteins at the active promoter relative to control samples (Storey-q < 0.05, fold change > 1.2). These proteins were enriched for transcription factors (TFs) and components of the spliceosome. To validate our candidate transcriptional regulators, we utilized computationally predicted TF binding and the >200 ChIP-Seq experiments performed in HEK293 cells by Encyclopedia of DNA Elements. In addition to validating dozens of candidate TFs as binders of the targeted genomic locus, we newly identify IRF2BP2 and glucocorticoid signaling as negative regulators of FOXP2 transcription, suggesting they each play a key role in FOXP2 gene expression. We further demonstrate that MS detects approximately one third of both binders and nonbinders, with more highly expressed genes significantly more likely to be detected regardless of binding status or locus specificity. Encyclopedia of DNA Elements ChIP-Seq binders not detected by MS show significantly lower expression compared to nonbinders only at the targeted FOXP2 promoter and not at off target loci.